Long-range goals of this project relate to the basic mechanisms underlying the action of abused drugs. The immediate focus is on nicotine; dependence on nicotine by tens of millions of cigarette smokers, with its associated health hazards, is an extraordinarily widespread and costly form of drug abuse. The proposed work deals in new ways with the effect of nicotine on the structure and function of CNS neurons. Two hypotheses, suggested by recent observations, will be investigated: (1) By interfering with the physiological control of neuronal nicotinic ACh receptors, nicotine may significantly perturb receptor concentration or position; (2) By excessively stimulating receptor-mediated ion flux, nicotine may cause Ca++ activated proteolytic damage to the structure of neuronal synapses. The specific experiments proposed are designed to investigate the normal cell biology of neuronal nicotinic receptors in addition to testing the effects of nicotine. Cells from the chick retina will be used because of their high level of receptors as well as their convenience for biochemical, cellular and developmental experiments. Two novel approaches will be used in these experiments. First, methods have been developed for isolating highly arborized neurons in order to identify which cells have nicotinic receptors and to determine where on those cells receptors are localized. Second, methods have been developed for examining isolated neurons with the high voltage electron microscope; this technique permits three-dimensional ultrastructural analysis of selected subcellular regions with remarkable resolution, clarity and speed. Overall, the specific experiments posed should help identify which cells in the CNS are sensitive to nicotine, provide new insight into the assembly of neuronal postsynaptic cholinergic membranes, and establish how cholinergic drugs such as nicotine might perturb this ongoing process. Such perturbations could have lasting effects on neuron structure and function well after the drugs are withdrawn.